DE2656155A1 - MALEINIMIDOBENZOIC ACID-N-HYDROXYSUCCINIMIDESTER AND ITS USE IN THE ENZYME MARKING OF ANTIGENS - Google Patents

Description

"Maleimidobenzoic acid N-hydroxysuccinimide ester and its" ver .. application for enzyme labeling of antigens "

Priority: December 12, 1975, Japan, No. 148 787/1975

The invention relates to maleimidobenzoic acid N-hydroxysuccinimide ester (hereinafter also abbreviated to MBS) the general

Formula I. ·

COO-N

(D

These compounds are valuable reagents for binding enzymes to antigens. The invention thus also relates to enzyme-labeled Antigens of the general formula II

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ΌΟΝΗ-

(II)

in which X and Y have different meanings and each have a Mean enzyme or antigen. Finally, the invention also relates to an enzyme immunological method using the aforementioned enzyme-labeled antigens and test packs containing the enzyme-labeled antigens.

Enzyme immunological processes are to be understood as meaning all processes where enzyme-labeled antigens for antigen-antibody reactions can be used.

The enzyme immunological method according to the invention is generally carried out in such a way that an enzyme-labeled antigen, an unlabelled antigen (i.e. the substance to be determined) and an antibody in a buffer solution of a competitive Subjects the antigen-antibody reaction, the enzyme-labeled, antigen bound to the antibody separates and the free enzyme-labeled antigen (i.e., enzyme-labeled. antigen to which no antibody is bound) and the amount of labeled antigen (i.e. the substance to be determined) from the enzymatic Activity of the enzyme-labeled antigen bound to the antibody or of the free enzyme-labeled antigen is determined.

Such methods are known per se and are, for example, in

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U.S. Patents 3,654,090, 3,839,153, and 3,850,752.

However, the known enzyme-labeled antigens used to perform enzyme immunological determinations certain disadvantages. For example, the conventional enzyme-labeled antigens are prepared by adding the enzyme and the antigen binds to each other using the following non-selective, bifunctional binding agents:

COOH COOH JJO, '~

(CH ₂ )

CHO CHO

O = C = N

iT = C = O

O ₂ Cl

SO ₂ Cl

SO ₂ Cl

These binding agents contain two identical functional groups, so that they are undesirable when they are used to bind enzyme and antigen By-products such as antigen-antigen complexes and / or enzyme-enzyme complexes in addition to the desired enzyme-antigen complex (i.e. the enzyme-labeled antigen). Accordingly, it is difficult to obtain the desired enzyme-labeled antigen from the mixture isolate these three products. In particular, the presence of enzyme-enzyme complexes causes interference in enzyme immunological Provisions.

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The object of the invention is to provide improved binding agents with which enzymes and antigens selectively can be bound to each other.

It was found according to the invention that the esters of the general Formula I are able to bind enzymes and antigens selectively to one another under very mild conditions. Furthermore, was found that enzyme-labeled antigens produced using MBS perform well in enzyme immunological determinations let use. Finally, using these enzyme-labeled antigens, valuable test packs can also be used for Establish implementation of enzyme immunological determination procedures.

According to the invention, enzymes and antigens can be selectively bound to one another using MBS of the general formula I. In this way, the desired enzyme-labeled antigens are obtained. In the literature, compounds are described, which are structurally related to MBS (cf. Helvetica Chimica Acta, Vol. 58, (1975), pp. 53I to 541). However, in this one Parts of the literature the use of these compounds for enzyme immunological Procedure not described.

To establish a bond between an enzyme and an antigen, the following reactions are carried out according to the invention: (a) Reaction of an antigen or enzyme which contains an amino group but no mercapto groups with the ester residue of MBS

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with formation of a product of the general formula III

CONH

(III)

in which X has the preceding meaning.

(b) Reaction of the product of the general formula III with an enzyme or antigen which contains a mercapto group and optionally also has amino groups, the maleimido radical of MBS in the product of the general formula III undergoes an addition reaction with the enzyme or antigen Formation of the enzyme-labeled antigen of the general formula II is subject.

Thus, MBS is an excellent bifunctional binding agent of high selectivity, with which enzymes and antigens in a two-stage reaction under very mild conditions can be bound. The binding agent according to the invention differs from the conventional ones in its binding effect Binding agents.

As the antigen to be bound to the enzyme, the same substance to be measured is generally used. Therefore come a number of substances from high molecular weight up too low molecular weight in question, for example the so-called "haptens".

The antigen used in the first step (a) does not contain any

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Mercapto group, but has an amino group or a group convertible into an amino group. The amino group can already be originally contained in the antigen or it can also be introduced chemically afterwards. Specific examples of antigens that originally have an amino group but no mercapto group are hormones such as angiotensin I, insulin, iodothyronine and chorionic gonadotropin (HCG), compounds with an amino group, some of which are similar in their chemical structure to the compound to be measured, such as α, α ¹ -diphenylpropylamine when measuring diphenylhydantoin, physiological amines such as serotonin, enzymes with the exception of the enzyme used to mark the antigen, virus-specific antigens such as virus hepatitis B, tumor antigens such as Oc-S ¹ otoprotein, immunoglobulins, like IgG and IgE. Examples of antigens that do not contain a ttercapto group, but into which an amino group can be introduced, are haptens, such as steroid hormones (eg estradiol).

All enzymes can be used in the first reaction stage (a) which have an amino group but no mercapto group. It is preferred to use enzymes which satisfy all or some of the following conditions:

(1) High substrate specificity

(2) High stability under the determination and storage conditions

(3) High solubility

(4) Simple, sensitive, quick and cheap determination

procedure

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(5) - absence of biological fluids

(6) There are no substrates, inhibitors and disruptive factors from biological fluids. "

(7) Me biological activity remains after the chemical conversion obtained for binding with the antigen.

Specific examples of such enzymes are peroxidase, glucose oxidase, alkaline phosphatase and the like.

All enzymes can be used in the second reaction stage (b) containing a mercapto group. Preferably, these enzymes will satisfy all or some of the compounds listed above. An example of this is ß-D-galactosidase. Other enzymes that incorporate a mercapto group can also be used is introduced. The introduction of a mercapto group into enzymes can be carried out, for example, according to the procedure in Archives of Biochemistry and Biophysics, Vol. 96 (1962), pp. 605 "to 612.

Furthermore, antigens can be used in the second stage (b) which originally already have a mercapto group. It but it is also possible to use antigens without mercapto groups into which such a group is introduced by chemical means leaves. The introduction of mercapto groups into protein or peptide antigens can be carried out according to the method given above carry out. In the case of antigens with -S-S bonds in the molecule, such as insulin, the mercapto group can be removed by reducing the -S-S bond

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are formed.

According to a preferred embodiment of the invention an antigen that has an amino group but not a mercapto group contains, bound to MBS in the first stage. Subsequently, the product obtained is in the second stage using an enzyme tied to a mercapto group.

In particular, it becomes an antigen of comparatively high molecular weight like insulin or angiotensin, bound to MBS in the first stage. The product obtained is then ß-D-galactosidase, which contains a Merc apt ο group, bound.

According to a particularly preferred embodiment, of the three positional isomers of the compounds of the general formula I, i.e. the o-substituted compound (o-MBS), the m-substituted Compound (m-MBS) und.the p-substituted compound (p-MBS), m-MBS is used because it is characterized by a special stability and is excellent in reactivity with the amino group. Furthermore, according to this particularly preferred embodiment an antigen with a comparatively high molecular weight, such as insulin or angiotensin, initially to m-MBS bound, the product obtained being bound to β-D-galactosidase, which originally contains a mercapto group.

The reaction according to the first and second stages can be carried out by placing the components in a buffer (pH

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6 to 8), bringing at 10 to 40 ⁰ C, preferably 20 to 3O ⁰ C, 10 to 180 minutes, optionally with stirring into contact. This reaction is preferably carried out in the presence of a small amount of a water-soluble organic solvent such as tetrahydrofuran, dioxane, acetone or ethanol, but care must be taken that the organic solvent does not inactivate one of the components, such as the enzyme or is denatured. The reaction product obtained in this way can easily be purified by customary methods, for example by washing with a buffer solution, column chromatography on three-dimensionally crosslinked dextran (Sephadex; Pharmacia Fine Chemicals), membrane filtration (for example Diaflo, Amicon Corp.).

The ratio of enzyme to antigen in the enzyme-labeled antigen of the general formula II, which is obtained in this way, depends on the type of enzyme or antigen and in particular on the number of amino or mercapto groups in the enzyme or Antigen.

The compounds of the general formula I can be easily prepared by adding a maleimidobenzoic acid of the general formula formula

ooff

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with N-hydroxysuccinimide of the formula

COURT

"at room temperature for 2 to 3 hours in an organic solvent, such as tetrahydrofuran, dioxane, benzene or acetone, in the presence of a dehydrating agent such as dicyclohexylcarbο-diimide, implements.

The antibody to the enzyme-labeled antigen can be immunized of corresponding animals, such as rabbits, horses, goats, guinea pigs or cattle, with the corresponding antigen, the contains an adjuvant. The antibody is formed in the serum; The antibody-containing Serum can be used directly, i.e. without further purification, for enzyme immunological determination. But it can also be done beforehand be subjected to cleaning. Furthermore, antibodies (antisera) against haptens can be produced by attaching the haptens a high molecular substance such as albumin, absorbs or binds and then the animal with the product obtained together with immunized with an adjuvant in a corresponding manner.

The enzyme immunological determination using the inventive enzyme-labeled antigen can be prepared, for example, as described in US Pat. Nos. 3,654,090, 3,850,752 and 3,839,153 be performed. This creates a competitive immune response

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-in

the enzyme-labeled antigen and one unlabeled Antigen, i.e. the substance to be determined, carried out in relation to the antibody in a buffer solution. Afterward the resulting enzyme-labeled antigen-antibody complex becomes separated by centrifugation. Another possibility is to precipitate the reaction mixture obtained above add a second antibody to the enzyme-labeled antigen-antibody complex (so-called "double antibody method") · Then the activity of the labeled enzyme in the precipitate or in the supernatant liquid to conventional Measured way. Finally, the amount of unlabeled antigen in the sample is calculated using a calibration curve is taken as a basis, which is used in the corresponding enzyme-immunological determination using a predetermined amount a standardized antigen. The second antibody can also be produced in the manner described above, wherein the first antibody (immunoglobulins) as an antigen for the production of the second antibody in the serum of an animal, which was not used to produce the first antibody is used.

Thus, in the enzyme immunological determination using the labeled antigen according to the invention, the following essential components used:

(a) An enzyme-labeled antigen produced by binding an enzyme and an antigen using MBS.

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(b) An antibody against the antigen of (a).

(c) A substrate for the enzyme of (a). If necessary, the following reagents can be used:

(d) A second antibody.

(e) A reagent different from (c) for measuring the enzyme activity.

The reagents (a), (b) and (d) can be kept cool in the form of a buffer solution. However, they are preferably used in Stored freeze-dried and dissolved in water or buffer before use. The freeze-dried Product can be produced by adding an appropriate solution, optionally with stabilizers, fillers or the like. Has been subjected to freeze-drying. Storage in the freeze-dried state is in terms of stability and easy handling particularly preferred.

The nature of the reagent (e) depends on the type of enzyme of (a) and the particular method. Examples are chromogenic reagents, coenzymes and enzymatic disruption agents Reaction.

The examples illustrate the invention.

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Example 1 Production of ο-, m- and p-MBS

A solution of 217 g of o-, m- or p-maleimidobenzoic acid in 30 ml of tetrahydrofuran are mixed with 130 mg of N-hydroxysuccinimide and 224 mg of dicyclohexylcarbodiimide. The mixture is 2 Stirred for hours at room temperature. The precipitated Ν, Ν'-dicyclohexylurea is filtered off. The filtrate is concentrated under reduced pressure. The residue obtained in this way is purified by column chromatography on silica gel using chloroform as the eluent. Afterward is the product of a mixture of diethyl ether and dichloromethane recrystallized. O-, m- or p-MBS with the properties given in Table I are obtained.

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Table I.

• . '· El ement aranalys e 10 ^N 2 ° 6th N 8th 92 IR spectrum (= C-H) 1495, ..... , 1446, ρ 1513, _f 1712 ' ( -CH ₀ CH ₀ - N M R spectra 3> Ha R ' 3H • iv ■■ " ^C 15 ^H - '·. (% ) (KBr) 1731, 1065 1075 1075 Suc- 6OMHz.CDCl IH ( ⁰ C) _. ,, £. ber ) 8th, 8 y 85 v> max (cm ~ (Maleimide 'l720. 699 f 832 ,. -COONO / S-'value Ha— <T / -CO- - found cinimide, , Suc- (= C-H) 1392, -CH-CH- H 8th, 1559, -COONO , , 1740, (-COONC) / 2.82 (s) Ha Hb C. 3100 1202, 1390, Once imid _% 4H 7.28-7.95 (m), 125 3.21 1770, 987, (-COONO / cinimid, .8, .13-8.37 (πι) / I. 57.33 N 92 3110 842, 701 1605 1718 6.86 (s) Hydrogen in "benzene R. Hb 2H 0-MBS 128 2.95 1773 (= C-H) 1208, Suc- 2H ring- '■ "■■ · 2H 57.29 8th? 72 , 1738, 1002 -COONC) / Ha- L \ - CO- Maleimide, ·. 1375, / - \ H ⁸ y cinimid, (-COONO; 2.90 (s) Ha Hb C. 1487 .6.99 ... ' 4H .7.57-7.77 (m), 182 3.21 1204, 8.03-8.23 (m), I. 57.33 N 92 830, 6 ^ 89 (s) Ha: Ha Hb 8Hz, 2H m-MBS 185 2.98 3095 2H Hb: 8Hz ,. 2ϊ \ 57.22 65 1774 R— L /} —CO- / \ H 2.90 (s) Ha Hb C. 4H 7.65 (d), J = 8, 198 3.21 8.23 (d).,. J = 8, ί: 57.33 6.90 (s) Ha: p-MBS 200 3.18 2H Hb: 56.90 Ha: Hb:

R: maleimide

JM

Of the two functional groups in these MBS compounds the maleimido group is to be regarded as unstable, while the Ester group has a lower reactivity for the antigen or the enzyme. The stability and reactivity of MBS was investigated. The results are summarized in Tables II and III.

Table II
MBS stability

Ver. . 30th incubation period 8th, 0 20 min 5 ..binding PH-
5.0 min ".. 69 0 7, 8th 3.1 'Area
6.0 7.0 43, 8th 18 4th o-MBS 2.9 6.2 21.4 52, 0 9, 5 m-MBS 3.8 2.5 7.1 37, p-MBS 6.6 32.0

Note: The numerical value in the table above gives the percentage on decomposed maleimido groups in MBS. Included 10 mmol of the compound to be investigated are dissolved in 20 ^ uI tetrahydrofuran and thoroughly with 0.5 ml a 0.05 M phosphate buffer of pH 6.0, 7.0, 7.5 or 8.0 or a 0.05 m citrate buffer with a pH of 5.0. The mixture is 20 and 30 minutes, respectively incubated.

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Zl

Table III Reactivity of MBS

link Reagent: lysine o-MBS 32.6 m-MBS 4-1.0 p-MBS 27.5

Note: The numerical values in the table above indicate the percentage of acylation of lysine with MBS. 1 mmol of the compound to be investigated is dissolved in 10 ^ uI tetrahydrofuran and mixed with 0.1 ml of a 0.1 M lysine solution in 0.05 M phosphate buffer with a ρH value of 7.5 . The mixture is reacted for 20 minutes at 3O ⁰ C.

Example 2 Determination of insulin
a) Binding of m-MBS to insulin

1 ml of 0.05 M phosphate buffer of pH 7 * 0 with a content of 6 mg (1 ^ uMoI) porcine insulin (25.5 U / mg, product of Schwarz / Mann company) is 75 ^ 1 of a solution of m-MBS

in tetrahydrofuran

(1.2; µmoles, i.e. 5 mg / ml) / added. The mixture is 30 minutes left to stand at room temperature and occasionally stirred. The mixture obtained is mixed with 1 ml of 1 M citrate-phosphate buffer added from pH 5.0. The precipitate formed is centrifuged at 800 g for 15 minutes.

The precipitate is then treated twice with 0.01 M citrate buffer

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(pH 5-3i 2 ml) and washed under reduced pressure dried. 5.5 mg / S-MBSj7 - / "~ Insulin_7 are obtained.

b) Binding of / m-MBS / ⁷ - / """Insulin " J to β-D-galactosidase

1 ml of 0.05 M phosphate buffer with a pH value of 7> 0 with a content

(0.93 nmol)
of 500 ^ g / ß-D-galactosidase from Escherichia coli (Boehringer Mannheim) is mixed with 0.15 ml of 0.05 m. phosphate buffer with a pH of 7.0 with a content of 15 l ^ ug (maleimide content 3.6 nmol) / ^ - MBS7- / f "liisulin_7 ', which has been obtained according to a), added. The mixture is left to stand for 2 hours at room temperature.

The resulting mixture is then poured onto a Sepharose 6B packed column of dimensions 1.8 χ 33 cm placed on top and with 0.01 m WaCl- - 0.02 m phosphate buffer of pH 7.0 eluted.

The desired ^ -D-Galactosidasj ^ - ^ - MB ^ T ^ / ^ Insu- HnZ- product is separated from free enzyme and from / S-MBS / ^r - / ~ Insuliii7 ^r . The yield is 80 percent, calculated from the enzymatic activity. The molar ratio of insulin to ß-D-galactosidase in the product is about 1: 1.8.

c) Insulin determination

The main fraction of the ZB-D-Galactosidas ^ T-Zm-MBST-Z-inguXin ^.

Product (ie the enzyme-labeled antigen) obtained according to b) is diluted 200-fold with water. 10 ) iL of the diluted enzyme-labeled antigen

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with 0.2 ml of 0.02 M phosphate buffer with a pH value of 7.0 (0.1 percent rabbit serum albumin, 1 millimolar MgClo, 0.1 M NaCl, 0.1 percent NaNO, the insulin (i.e. unlabelled antigen, O up to 20 ^ uU) and 50 ) ti. Anti-pig insulin sea pig anti serum (Dainabot Radioisotope Lab. Ltd., Japan; ie first antibody) contains. The mixture is allowed to stand 16 hours at 4 ⁰ C and then with 10 ^ 1 anti-rabbit 7s / '- globulin anti-guinea pig serum (ie, the second antibody) was added. The mixture is left to stand for a further 8 hours at 4 ^° C. and then centrifuged at 800 g for 15 minutes. The β-D-galactosidase activity in the supernatant liquid or in the precipitate is measured according to the method explained in d). The calibration curve shown in FIG. 1 is obtained from the values. According to this enzyme-immunological method, insulin can thus be _{measured in amounts of 0.5 to 20 ×} IiIr.

d) Measurement of the β-D-galactosidase activity

0.15 ml of a substrate solution (0.1 millimolar 4-methylumbelliferyl-ß-D-galactoside, 0.02 m. Sodium phosphate, 0.1 percent rabbit serum albumin, 1 millimolar MgClp, 0.1 m NaCl, 0.1 percent NaN, , pH 7.0) are mixed with 50 ^ uI of the supernatant liquid which has been obtained after the antigen-antibody reaction of c). The mixture is left to stand at 30 ^° C. for 60 minutes.

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Another possibility is to wash the precipitate obtained according to c) with 2 ml of 0.05 .mu.m phosphate buffer with a pH of 7 ₅ O and to add 0.15 ml of the above substrate solution. The mixture is left to stand at 30 ^° C. for 30 minutes.

The above mixtures are each with 2.5 ml of a 0.1 m glycine-NaOH buffer solution of pH 10.3 added, to stop the reaction. The 4-methylumbelliferone formed in the reaction mixture depending on the activity of the enzyme-labeled antigen is performed with an MPF-4 spectrofluorometer (Hitachi, Ltd., Japan) at an excitation wavelength of 365 nm and measured at an emission wavelength of 448 nm.

Example 3 Measurement of Angiotensin I
a) Binding of m-MBS to angiotensin I.

1 ml of 0.5 M phosphate buffer with a pH value of 7 »0 with a content of 2 mg (1.4 _/ µmol) angiotensin I is added to 0.26 ml of a solution of ϊα-MBS (4.2, JoMoI, ie 5 mg / ml) in tetrahydrofuran. The mixture is left to stand at room temperature for 30 minutes, with occasional shaking. The reaction mixture is then mixed with 0.5 ml of 1M citrate buffer with a pH value of 5 »0 and then transferred to a three-dimensionally crosslinked! Dextran (Sephadex G-15) loaded column with dimensions of 1.9 x 38 cm. The elution becomes 0.02 m

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Citrate buffer with a content of 0.1 M NaGl carried out. The desired product / ^ - MBS7- / ~ Angiotensin I_7 separated from unreacted angiotensin I and m-MBS.

b) Binding of / m-MBST - / "angiotensin I 7 to BD-galactosidase 0.5 ml. of a 0.05 m phosphate buffer with a ρH value of 7» 0

. a content of 500 ^ Ug (0.93 nmol) ß-D-galactosidase are mixed with 0.5 ml 0.05 M phosphate buffer with a pH value of 7.0 _? which contains the / ff-MBS7- / ~ angiotensin I_7 (maleimido content: 2.8 nmol) obtained according to a). The mixture is left to stand at room temperature for 2 hours. It is then placed on a column of dimensions 1.8 × 4-2 cm packed with three-dimensional crosslinked dextran (Sephadex G-25) and eluted with 0.02 M phosphate buffer containing 0.1 M NaCl. A main fraction is obtained with a content of / l "-D-galcatosidase7- / m-MBS7- / ~ angiotensin I_7.

c) Measurement of angiotensin I

The main fraction with a content of / E- D-GaIactosidas_e_7- ^ ü-MBS_7- / ~ Angiotensin I_7, ie the enzyme-labeled antigen obtained according to b), is diluted 1000 times with water. 10 μl of diluted, enzyme-labeled antigen is mixed with 0.2 ml of the buffer from Example 2 c) containing 0 to 800 ng (ng = nanograms) of angiotensin I, ie unlabelled antigen, and 50 μl of anti-angiotensin I rabbit serum, ie first antibody, contains, mixed. The mixture is left to stand for 8 hours at room temperature and then 10 μl of anti-

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Rabbit 7s / 'globulin goat antiserum, ie second antibody, added. The mixture is then left to stand at 4 ^° C. for 16 hours and then centrifuged at 800 g for 15 minutes. The enzyme activity of the supernatant liquid and of the precipitate obtained is determined according to Example 2 d). This gives the calibration curve shown in FIG. 2 for the enzyme-immunological determination of angiotensin

I. According to this method, angiotensin can be converted into

-12

Determine quantities from 5 to 100 pg (pg = picogram = 10 "g).

Example 4

Measurement of triiodothyronine (abbreviated "GV ' a) Production of / ~ T ^ 7- / m-, P- or o-MBS7- / ~ ß-D-galactoside 7

50 μl of a 0.1 μm aqueous NaOH solution with a content of 0.5 μMoI T ₃ (Sigma Chemical Co.) are mixed with 1 ml of 0.05 M phosphate buffer with a pH of 7-0. The solution obtained is added dropwise with stirring with 25 ^ l of a solution of 0.5 / iMol m-, p- or o-MBS in tetrahydrofuran. The mixture is reacted for 10 minutes at room temperature and then 40 μl of 0.2 M citrate-phosphate buffer with a pH of 4.3 are added. The precipitate obtained is centrifuged off for 10 minutes at 800 g and washed with 1 ml of 0.05 M citrate-phosphate buffer of pH 4.3 by centrifugation. The precipitate obtained in this way is mixed with 1 ml of 0.05 ei phosphate buffer with a pH of 7.0 and then 100 (0.19 nmoles) of β-D-galactosidase are added while stirring. After 10 minutes

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The mixture is stirred with 5 ml of a 0.1 M glycine-NaOH buffer added from pH 10.3. The mixture is filtered through Diaflo PM 30 (Amicon Corp.) to remove any unreacted / m-, p- or o-MBS_7- / ~ T; z_7 or unconverted T ^ to remove. The precipitate obtained is also washed twice with 0.05 M phosphate buffer of pH 7.0 (3 ml) and then added to 1 ml of 0.05 M phosphate buffer with a pH of 7.0. Then with 0.5 ml of 1 percent bovine erumalbumin solution added.

b) Measurement of T ^

The fraction of / ~ T; z Tv ^ ä-, p- or o-MBS7- / ß-D-galactosidase7 obtained according to a), ie the enzyme-labeled antigen, is diluted 100 times with water. 10 ^ uI of diluted, enzyme-labeled antigen are mixed with 1 ml of a 0.02 M tris-HCl solution containing 0.9 percent NaCl at pH 7.0, the 0 to 50 ng standard T, and 50 µl of T ^ rabbit antiserum (Dainippon Pharmaceutical Co., Ltd.) diluted 100 times with water is added. The mixture will. Left to stand at 5 ° C for 5 hours. 10 ₇ JiI anti-rabbit IgG goat antiserum (Miles Laboratories Inc.) is then added. The mixture is left to stand at 5 ^° C. for 16 hours and then centrifuged at 800 g for 10 minutes. 1 ml of the above buffer is added to the precipitate obtained. Then the mixture continues

centrifuged. The precipitate is 0.15 ml with 0.1 millimolar 4-methylumbelliferyl-ß-D-galactoside - 0.02 m phos-

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phate buffer of pH 7.0 added. The mixture is 30 minutes, "at 30 ⁰ C allowed to stand. Then, the ß-D-GaIactosidase activity is described in Example 2 d)" is determined. The calibration curve shown in FIG. 3 is thereby obtained. This means that 200 pg to 50 ng T ₇ can be determined using this enzyme-immunological "method.

Example 5

a) Production of / 0) iphenylpropylamine7 - / ^ - MBS'7- / ß "'- D-galactosi-

that7

A solution of 3.2 µMoI (1.1 mg / 0.16 ml) of m-MBS in tetrahydrofuran is with a solution of 3.2 ^ uMoI 3,3-diphenylpropylamine added in 32 ^ 1 ethanol. The mixture is 1 Stirred for one hour at room temperature. Then 25 / ul of the reaction mixture with 0.05 M phosphate buffer of pH 7.0 with a content of 100 ^ uig (0.19 nmol) ß-D-galactosidase touched. The mixture is allowed to stand for 10 minutes and then filtered through Diaflo EM 30 (Amicon Corp.). Of the The residue is reduced to pH twice with 0.05 M phosphate buffer 7.0 (3 ml) and filtered. The residue obtained in this way is adjusted to pH with 1 ml of 0.05 M phosphate buffer 7.0 and 0.5 ml of 1% beef serum albumin solution were added.

b) measurement; of diphenylhydantoin according to the enzyme immunological method

10 ia1 of that obtained according to a), diluted 10 times

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Solution of ^ iphenylpropylamin7 - ^ - MBSy - / ^ - D-Galactosidasj37, 0 to 50 ng of diphenylhydantoin and 10 µl of diphenylacetylated bovine albumin rabbit antiserum diluted 20 times with water (Dainippon Pharmaceutical Co., Ltd.) become 1 ml of a 0.02 m tris-HCT solution with a content of 0.9 percent HaGl with a pH value of 7 ₅ O is given. The mixture is left to stand at 5 ^° C. for 5 hours. 10 ^ l of goat anti-rabbit IgG antiserum are then added. The mixture is left to stand at 5 ° ^{C. for} 16 hours and then centrifuged at 800 g for 10 minutes. The obtained precipitate is washed with 1 ml of the above buffer while centrifuging. washed. The β-D-galactosidase activity is then measured according to Example 2 d). The calibration curve shown in FIG. 4 is obtained. This enzyme-immunological method can thus be used to determine 0.8 to 100 ng of diphenylhydantoin.

Example 6

Production of a test pack for measuring insulin A: buffer

60.6 g bovine serum albumin, 272.7 g NaCl, 14-9.5 g Na ₂ HPO ^ .12H

29.7 g of NaH ₂ PO ^ -H ₂ O and 30.3 g of NaN ₅ are mixed thoroughly. In each case 5.374 mg are filled into 100 brown 15 ml bottles. Before use, the contents of the bottle are made up to a final volume of 300 ml with water. The solution then has a content of 0.2 percent bovine serum albumin, 0.9 percent NaCl, 0.1 percent NaN, and 0.02 m phosphorus

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34

phate buffer (pH 7.0). (Reagent A).

B: enzyme-labeled antigen

0.53 ml of the fraction obtained according to Example 2 with a Content of ^ -D-Galactosidas will be £ 7 - / ^ - MBJ37- / ~ Insulin_7 reagent A (buffer) is added to a total volume of 1060 ml, thus a 2000-fold dilution takes place. 100 brown 15 ml bottles are each with 10.5 ml of this Mixture filled (reagent B).

C: first antibody

3O ₇ UI anti-porcine insulin guinea pig antiserum (37,000 units of the first antibody) prepared according to J. Clin. Invest., Vol. 39 0960), p. 1157, 222 ml of a 0.5 percent aqueous solution of a normal guinea pig serum are added and thus diluted about 7,000 times. 100 "brown 15 ml bottles are each mixed with 2.2 ml of this mixture and then freeze-dried.

In use, this freeze-dried product is dissolved in 11.0 ml of buffer. A solution of the first is obtained Antibody at a concentration of 1 U / 0.1 ml (reagent C).

1 U (unit) is the amount of antibody

125 those in the implementation of the antibody with 100 pg ^ J-insulin reacts with 50 percent of the insulin labeled with 125 J.

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D: Second antibody

2.1 ml each of anti-guinea pig IgG rabbit antiserum, manufactured according to Biochem. J., 88, 137 (1963) Filled into 115 ml brown bottles and freeze-dried.

In use, the freeze-dried product in 10.5 ^m l reagent A (buffer) (reagent D). 0.1 ml of this solution has sufficient activity that the antibody reaction takes place with 0.1 ml of reagent C (first antibody).

E: standard insulin

100 brown 15 ml bottles are each with 1.0 ml an insulin solution with a concentration of 640 ^ uU / ml filled and freeze-dried.

In use, the freeze-dried product is dissolved in ' ^: water to a final volume of 2.0 ml. This solution contains 320, UUZmI insulin. This insulin stock solution is diluted twice to create an insulin calibration curve (reagent E).

Q: substrate

63 mg ^ -Methylumbelliferyl-ß-D-galactoside, 8679 mg Na ₂ HPO ^. 0.176O mg NaH ₂ PO ^, 176O mg bovine serum albumin, 176O mg, 352 mg MgCl ₂ and 10.56 g NaCl are mixed thoroughly, 709825/1059

Each 227 ^ S of this mixture becomes 100 brown Given 20 ml bottles. When used, the product is dissolved with water to a final volume of 16.0 ml (reagent

G: buffer

In each case 20.0 ml of a 1.5 M glycine-NaOH buffer with a pH value 10.3 are filled into 100 brown "20 ml bottles. When used, add water to a final volume of 300 ml diluted (reagent G).

In this way, 100 test packs are produced, each containing 1 bottle of reagents A to G (hereinafter referred to as "Insulin EIA Test Pack"), which is for 100 determinations sufficient., received.

When using this insulin EIA test pack for the The enzyme-immunological determination of insulin is carried out as follows;

A mixture of 0.5. ml of reagent A (buffer), 0.1 ml of reagent B (enzyme-labeled antigen), 0.1 ml of reagent C (first antibody) and the sample to be examined or a corresponding amount of a standard insulin solution is stored for 24 hours at 4 ° C incubated. 0.1 ml of reagent D (second antibody) is added to the mixture obtained. The mixture is incubated for 8 hours at 4 ⁰ C and then centrifuged 15 minutes at 800th The resulting precipitate is with

70 9 825/1059

St.

2.0 ml of reagent A (buffer) washed with centrifugation. The enzyme activity is then measured according to Example 2 d).

Example 7

Measurement of insulin in human serum The insulin concentration in the sera of 54 test persons who are obviously healthy is determined using the insulin EIA test pack prepared according to Example 6 and a commercially available insulin test pack (insulin RIA test pack, trade name of Dainabot Radioisotope Lab. , Ltd., a test pack for measuring insulin using a radioimmunological method). The results are shown in FIG. 5. 5 shows that the insulin concentrations determined by both methods are well correlated (correlation coefficient = 0.92).

709825/1059

Claims (1)

Claims 1; Maleimidobenzoic acid N-hydroxysuccinimide ester of the general formula COO-N 2. m-isomer of the compounds of claim 1 having the following formula COO-N 3. Enzyme-labeled antigen, suitable for enzyme immunological Provisions, the general formula CONH- in which X and Y are each different and represent an enzyme or an antigen. . Enzyme-labeled antigen according to claim 3 * by the following general formula 709825/1059 ORSGiMAL fMSPECTED oöm- in which X and Y have the same meaning as in claim 3. 5. Enzyme-labeled antigen according to claim 3> characterized in that X is an antigen and Y is an enzyme. 6. Enzyme-labeled antigen according to claim 3 »characterized in that X is an enzyme and Y is an antigen is. 7. Enzyme-labeled antigen according to claim 3 »marked thereby ei.chnet that one of the residues X and Y is a hormone is. 8. Enzyme-labeled antigen according to claim 7 , characterized in that it contains insulin, angiotensin I or iodothyronine as a hormone. 9. Enzyme-labeled antigen according to Claim.3? through this marked that one of the radicals X and Y is a hapten. 10. Enzyme-labeled antigen according to claim 9 »characterized gekenn'ζ eichnet that the hapten partially the 709825/1059 has the same structure as the substance to be measured. 11. Enzyme-labeled antigen according to claim 10, characterized in that it is a hapten diphenylpropylamine contains. 12. Enzyme-labeled antigen according to claim 3, characterized in that Y is ß-D-galactosidase. 1Y. Enzyme-labeled antigen according to claim 3 »thereby marked that X is peroxidase, glucose oxidase or alkaline phosphatase. 14. Enzyme-labeled antigen according to claim 5? through this characterized in that X is insulin and Y is β-D-galactosidase. 15 · Enzyme-labeled antigen according to claim 5i thereby marked that X triiodothyronine and Y ß-D-galactosidase is. 16. Enzyme-labeled antigen according to claim 5 »thereby marked that X angiotensin I and Y ß-D-galactosidase is. 17 · Enzyme immunological method of determination, thereby characterized in that one is an enzyme-labeled 709825/1059 Antigen of the general formula COHH used in which X and Y are different and each represent an enzyme or antigen, ". 18. Test pack for enzyme-immunological determination procedures, geke nn is characterized by the following three components as essential components:
Component A: enzyme-labeled antigen of the general formula com in which -X and Y are different and each represent an enzyme or antigen, Component B: an antibody against the component's antigen A and Component C: a substrate for the component A enzyme. 19 test pack according to claim 18, characterized in that that component A is an enzyme-labeled antigen of the general formula 709825/1059 β-D-fractalactosidase 0ONH •Insulin 20. Process for the preparation of an enzyme-labeled antigen for enzyme-immunological determination methods of the general
Formula II C (Mi di) in which X and Y are different and in each case an enzyme or
Antigen, characterized in that a compound of the general formula I COO-N (I) with an antigen or enzyme which contains an amino group but no mercapto group to form a product of the general formula III implements com (in) 709825/1059 in which X has the above meaning, and the product of the general formula III with an enzyme or antigen with a Reacts mercapto group. 21. Procedure according to. Claim 20, characterized in that that the compound of general formula I with an antigen that has an amino group but no mercapto group contains, reacts and the product of the general formula III obtained with an enzyme which has a mercapto group contains, implements. 709825/1059